Quantification of adaptive immune cell genomes in a complex mixture of cells

Abstract

Compositions and methods are described for highly sensitive quantification of the relative representation of DNA from adaptive immune cells (e.g., T and/or B lymphocytes) in DNA extracted from complex mixtures of cells that include cells which are not adaptive immune cells. Included are methods for determining the relative presence in a tumor of tumor infiltrating lymphocytes (TIL), the relative presence of lymphocytes infiltrating a somatic tissue that is the target of an autoimmune disease, and the relative presence of lymphocytes infiltrating a transplanted organ.

The invention claimed is:  
     
       1. A method for determining a relative quantity of tumor-infiltrating lymphocytes in a solid tumor, comprising:
 (a) obtaining a sample comprising a solid tumor tissue; 
 (b) amplifying by PCR at least 80% of all rearranged TCR or Ig CDR3-encoding regions present in said sample using a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs: 1-52, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, and 843-879 and a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of 53-63, 65, 215-220, and 247 to produce a plurality of rearranged DNA amplicons; 
 (c) amplifying by PCR a control sequence present in said sample using a pair of control sequence primers, wherein said control sequence primers are capable of amplifying a control sequence that is not an adaptive immune receptor gene and present in all cells in said sample; 
 (d) quantifying a number of adaptive immune receptor sequence reads in said sample generated from high-throughput sequencing (HTS) of said plurality of rearranged DNA amplicons; 
 (e) quantifying a number of control sequence reads in said sample using said amplified control sequence; and 
 (f) comparing said number of adaptive immune receptor sequence reads and said number of control sequence reads to estimate a relative quantity of tumor-infiltrating lymphocytes in said solid tumor. 
 
     
     
       2. The method of  claim 1 , further comprising quantifying a number of unique sequence reads generated from said HTS, wherein each unique sequence read comprises a sequence distinct from the other sequence reads. 
     
     
       3. The method of  claim 1 , wherein said number of control sequence reads represents a total number of diploid genomes in said sample. 
     
     
       4. The method of  claim 3 , wherein said comparing comprises dividing said number of control sequence reads in half and determining a ratio between said number of adaptive immune receptor sequence reads and half of said number of control sequence reads. 
     
     
       5. The method of  claim 1 , wherein said comparing comprises estimating a total number of adaptive immune cells in said sample by dividing said number of adaptive immune receptor sequence reads by a numerical factor. 
     
     
       6. The method of  claim 1 , wherein said plurality of rearranged DNA amplicons comprises at least 10 6  DNA molecules. 
     
     
       7. The method of  claim 1 , wherein said plurality of rearranged DNA amplicons comprises at least 10 5  DNA molecules. 
     
     
       8. The method of  claim 1 , wherein each V-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR or Ig V-encoding gene segment, and wherein said V-segment oligonucleotide primers specifically hybridize to at least 80% of all functional TCR or Ig V-encoding gene segments that are present in said sample. 
     
     
       9. The method of  claim 1 , wherein each J-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR or Ig J-encoding gene segment, and wherein said J-segment oligonucleotide primers specifically hybridize to at least 80% of all functional TCR or Ig J-encoding gene segments that are present in said test sample. 
     
     
       10. The method of  claim 1 , wherein each amplified rearranged TCR or Ig CDR3-encoding region is less than 600 nucleotides in length. 
     
     
       11. The method of  claim 1 , wherein each of said rearranged TCR or Ig CDR3-encoding regions encode a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide comprising a V gene recombination signal sequence (RSS) and a T cell receptor (TCR) J-region polypeptide or an immunoglobulin (Ig) J-region polypeptide comprising a J gene RSS, and wherein each rearranged DNA amplicon comprises (i) at least 10, 20, 30 or 40 contiguous nucleotides of a sense strand of a TCR or Ig V-encoding gene segment, said at least 10, 20, 30 or 40 contiguous nucleotides being situated 5′ to said V gene RSS and (ii) at least 10, 20 or 30 contiguous nucleotides of a sense strand of a TCR or Ig J-encoding gene segment, said at least 10, 20 or 30 contiguous nucleotides being situated 3′ to said J gene RSS. 
     
     
       12. The method of  claim 1 , wherein said tumor-infiltrating lymphocytes are T cells or B cells. 
     
     
       13. The method of  claim 1 , wherein said rearranged TCR or Ig CDR3-encoding regions are selected from the group consisting of rearranged TCRα CDR3-encoding regions, TCRβ CDR3-encoding regions, TCRγ CDR3-encoding regions, TCRδ CDR3-encoding regions, IgH CDR3-encoding regions, Igκ CDR3-encoding regions, and Igλ CDR3-encoding regions. 
     
     
       14. A method for quantifying a relative representation of tumor infiltrating T cells in a solid tissue tumor sample, comprising:
 obtaining DNA templates from said sample; 
 amplifying rearranged T cell receptor DNA molecules utilizing a plurality of V-segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOs: 1-52, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695 and 843-879 and a plurality of J-segment oligonucleotide primers comprising sequences selected from the group consisting of 53-63, 65, 215-220, and 247 in a single multiplex PCR from said DNA templates to produce a multiplicity of amplified rearranged DNA molecules; 
 sequencing said multiplicity of amplified rearranged DNA molecules by high-throughput sequencing (HTS) to produce rearranged T cell receptor sequence reads; 
 determining a number of rearranged T cell receptor DNA molecules from said rearranged T cell receptor sequence reads, wherein said number of T cell receptor DNA molecules is proportional to a number of T cells in said sample; 
 determining a number of diploid genomes in the sample, wherein said number of diploid genomes represents a number of total cells in the sample; and 
 quantifying a ratio of the relative representation of tumor infiltrating T cells in said sample by comparing said number of T cells by said number of total cells in the sample. 
 
     
     
       15. The method of  claim 14 , wherein each V-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR V-encoding gene segment, and wherein said V-segment oligonucleotide primers specifically hybridize to at least 80% of all functional TCR V-encoding gene segments that are present in said sample. 
     
     
       16. The method of  claim 15 , wherein each J-segment oligonucleotide primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional TCR J-encoding gene segment, and wherein said J-segment oligonucleotide primers specifically hybridize to at least 80% of all functional TCR J-encoding gene segments that are present in said test sample. 
     
     
       17. The method of  claim 14 , wherein each of said rearranged TCR encodes a T cell receptor (TCR) V-region polypeptide comprising a V gene recombination signal sequence (RSS) and a T cell receptor (TCR) J-region polypeptide comprising a J gene RSS, and wherein each rearranged DNA molecule comprises (i) at least 10, 20, 30 or 40 contiguous nucleotides of a sense strand of a TCR V-encoding gene segment, said at least 10, 20, 30 or 40 contiguous nucleotides being situated 5′ to said V gene RSS and (ii) at least 10, 20 or 30 contiguous nucleotides of a sense strand of a TCR J-encoding gene segment, said at least 10, 20 or 30 contiguous nucleotides being situated 3′ to said J gene RSS. 
     
     
       18. The method of  claim 14 , wherein said number of diploid genomes in said sample is determined by contacting said sample with a pair of control sequence primers and by amplifying a control sequence from said DNA templates, wherein said control sequence primers are capable of amplifying a control sequence present in all cells in said sample. 
     
     
       19. The method of  claim 14 , further comprising quantifying a number of unique sequence reads generated from said HTS, wherein each unique sequence read comprises a sequence distinct from the other sequence reads. 
     
     
       20. The method of  claim 14 , wherein said comparing comprises dividing said number of T cells by a numerical factor. 
     
     
       21. The method of  claim 14 , wherein said rearranged DNA molecules are selected from the group consisting of rearranged TCRα CDR3-encoding regions, TCRβ CDR3-encoding regions, TCRγ CDR3-encoding regions, and TCRδ CDR3-encoding regions. 
     
     
       22. The method of  claim 1 , wherein the amplifying steps of (b) and (c) are performed in the same PCR reaction. 
     
     
       23. The method of  claim 1 , wherein the amplifying steps of (b) and (c) are performed in separate PCR reactions.